Import and export of bacterial protein toxins.

The paper provides a short overview of three investigated bacterial protein toxins, colicin M (Cma) of Escherichia coli, pesticin (Pst) of Yersinia pestis and hemolysin (ShlAB) of Serratia marcescens. Cma and Pst are exceptional among colicins in that they kill bacteria by degrading the murein (peptidoglycan). Both are released into the medium and bind to specific receptor proteins in the outer membrane of sensitive E. coli cells. Subsequently they are translocated into the periplasm by an energy-consuming process using the proton motive force. For transmembrane translocation the colicins unfold and refold in the periplasm. In the case of Cma the FkpA peptidyl prolyl cis-trans isomerase/chaperone is required. ShlA is secreted and activated through ShlB in the outer membrane by a type Vb secretion mechanism.

CbrA is a flavin adenine dinucleotide protein that modifies the Escherichia coli outer membrane and confers specific resistance to Colicin M.

Colicin M (Cma) is a protein toxin produced by Escherichia coli that kills sensitive E. coli cells by inhibiting murein biosynthesis in the periplasm. Recombinant plasmids carrying cbrA (formerly yidS) strongly increased resistance of cells to Cma, whereas deletion of cbrA increased Cma sensitivity. Transcription of cbrA is positively controlled by the two-component CreBC system. A ΔcreB mutant was highly Cma sensitive because little CbrA was synthesized. Treatment of CbrA-overproducing cells by osmotic shock failed to render cells Cma sensitive because the cells were resistant to osmotic shock. In a natural environment with a growth-limiting nutrient supply, cells producing CbrA defend themselves against colicin M synthesized by competing cells. Isolated CbrA is a protein with noncovalently bound flavin adenine dinucleotide. Sequence comparison and structure prediction assign the closest relative of CbrA with a known crystal structure as digeranylgeranyl-glycerophospholipid reductase of Thermoplasma acidophilum. CbrA is found in Escherichia coli, Citrobacter, and Salmonella bongori but not in other enterobacteria. The next homologs with the highest identity (over 50%) are found in the anaerobic Clostridium botulinum group 1 and a few other Firmicutes.

Activation of colicin M by the FkpA prolyl cis-trans isomerase/chaperone.

Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA.

Import of periplasmic bacteriocins targeting the murein.

Colicins are the only proteins imported by Escherichia coli and thus serve as tools to study the protein import mechanism. Most of the colicins studied degrade DNA, 16S RNA or tRNA in the cytoplasm, or form pores in the cytoplasmic membrane. Two bacteriocins, Cma (colicin M) and Pst (pesticin), affect the murein structure in the periplasm. These two bacteriocins must be imported only across the outer membrane and therefore represent the simplest system for studying protein import. Cma can be reversibly translocated across the outer membrane. Cma and Pst unfold during import. The crystal structure of Pst reveals a phage T4L (T4 lysozyme) fold of the activity domain. Both bacteriocins require energy for import which is translocated from the cytoplasmic membrane into the outer membrane by the Ton system. Cma kills cells only when the periplasmic FkpA PPIase (peptidylprolyl cis-trans isomerase)/chaperone is present.

Mapping functional domains of colicin M.

Colicin M (Cma) lyses Escherichia coli cells by inhibiting murein biosynthesis through hydrolysis of the phosphate ester between C(55)-polyisoprenol and N-acetylmuramyl (MurNAc)-pentapeptide-GlcNAc in the periplasm. To identify Cma functional domains, we isolated 54 point mutants and small deletion mutants and examined their cytotoxicity levels. Activity and uptake mutants were distinguished by osmotic shock, which transfers Cma into the periplasm independent of the specific FhuA receptor and the Ton system. Deletion of the hydrophobic helix α1, which extends from the compact Cma structure, abolished interference with the antibiotic albomycin, which is transported across the outer membrane by the same system as Cma, thereby identifying α1 as the Cma site that binds to FhuA. Deletion of the C-terminal Lys-Arg strongly reduced Cma translocation across the outer membrane after binding to FhuA. Conversion of Asp226 to Glu, Asn, or Ala inactivated Cma. Asp226 is exposed at the Cma surface and is surrounded by Asp225, Asp229, His235, Tyr228, and Arg236; replacement of each with alanine inactivated Cma. We propose that Asp226 directly participates in phosphate ester hydrolysis and that the surrounding residues contribute to the active site. These residues are strongly conserved in Cma-like proteins of other species. Replacement of other conserved residues with alanine inactivated Cma; these mutations probably altered the Cma structure, as particularly apparent for mutants in the unique open ß-barrel of Cma, which were isolated in lower yields. Our results identify regions in Cma responsible for uptake and activity and support the concept of a three-domain arrangement of Cma.